Interactions between proteins and nucleic acids and nucleic acid conformations are commonly examined by “footprinting” methods. In these methods, a nucleic acid molecule is fragmented by applying an agent that produces nicks in the phosphodiester backbone of the molecule. Regions of the molecule devoid of nicks are then sought. Such regions devoid of nicks are regions that were protected from the effects of the agent. Such protection may be due, for example, to binding of a ligand to a specific sequence of bases in the nucleic acid or to the conformation of the molecule(1, 2). The prerequisite of such assays is the ability to produce and detect high-quality nucleic acid fragmentation. Nucleic acid fragmentation can be achieved by using a variety of enzymatic and chemical reagents(3). Another method of nucleic acid fragmentation referred to as “chemical hydroxyl radical footprinting” uses Fenton chemistry(4-6) and peroxonitrous acid(7). In this method, hydroxyl radicals (—OH) engender breaks of the phosphodiester backbone in a non-specific sequence manner. Using hydroxyl radical methods over enzymatic footprinting is advantageous because it provides great sensitivity to nucleic-acid structures, such as sequence-dependent curvature(8) and RNA folding(9).
Nucleic acid cleavage by hydroxyl radical is predominantly dependent upon the solvent accessibility of the phosphodiester backbone. Additionally, it is relatively insensitive to base sequence, and it is not important whether the nucleic acid is single or double stranded(10). —OH can be generated by Fe-EDTA catalysis or by γ-rays, β particles, fast neutrons, and X-ray radiation(11-13). The radiolysis of water by X-rays with energies from 100 eV up to the MeV range produces free electrons and —OH according to the overall reaction illustrated in equation (1)(14).

The —OH generated by this reaction can abstract a hydrogen from the C′4 carbon of the ribose sugar of DNA and RNA, leading to breakage of the phosphodiester backbone of the polymer(15). Controlled exposure of protein-nucleic acid complexes to X-rays has been used to detect specific interactions within such complexes(11, 16). X-ray mediated footprinting has been shown to be an attractive method for time-resolved footprinting studies, because it can produce a high flux of —OH that fragments the nucleic acid backbone in millisecond time scales with basepair resolution. The recent development of synchrotron time-resolved X-ray footprinting demonstrated the utilization of this method to study dynamic structural changes in RNA folding(17).
High radiation sources like synchrotron can produce a sufficiently high photon flux to generate a sufficient concentration of —OH radicals to fragment nucleic acids within of tenths of milliseconds(13). However, the use of a synchrotron radiation source in footprinting is limited due to its relative inaccessibility and the high cost of its operation.